Autoantibodies directed against the islet cells of the pancreas are indicators of ongoing beta cell destruction, predict preclinical disease, and identify susceptible individuals at risk for development of diabetes. Immunologic, genetic, and epidemiologic studies indicate that the majority of patients with insulin dependent diabetes mellitus (IDDM) have a long, clinically asymptomatic, prediabetic period. This phase is characterized by the presence of autoimmune phenomena and very subtle metabolic changes which antedate the clinical manifestations of insulin deficiency by many months or even years. Organ specific autoimmune reactions play an essential role in the development of progressive destruction of the insulin producing cells of the islets of Langerhans.
Type I diabetes is a chronic autoimmune disease with six major sequential stages (Eisenbarth, N. Engl. J. Med. 314: 136, 1986). Briefly, in genetically susceptible individuals (Stage I), an unidentified factor (Stage II) triggers the development of a variety of immunologic abnormalities (Stage III). These abnormalities precede a progressive loss of insulin secretion (Stage IV), until clinical diabetes becomes manifest (Stage V). At this point, some residual beta cell function persists, but almost complete destruction of the beta cells finally follows (Stage VI). Additionally, in patients with Type II (non-insulin dependent) and gestational diabetes, the presence of islet cell antibodies portends insulin deficiency and defines subgroups that have a slowly progressive form of Type I diabetes (Irvine et al., J. Clin Lab Immunol. 2: 23, 1979; Groop et al., Diabetes 35: 237, 1986; Steel et al., J. Lab. Clin. Immunol. 4: 83, 1980; DiMario et al., Diabetologia 25: 392, 1983; Tetsuro et al., Diabetes 36: 510, 1987; Gleichman et al., Diabetologia 27: 90, 1984). The rate of beta cell destruction is accelerated in patients with adult onset diabetes who have islet cell antibodies.
Islet Cell Antibodies (ICA) were originally detected by indirect immunofluorescence. Briefly, the patient serum is layered on an unfixed cryostat section of human pancreas from a blood group O donor. Binding of ICA to islet cells is then revealed with an antihuman IgG reagent, conjugated to fluorescein isothiocyanate, and detected with an ultraviolet microscope. If ICA are present in the serum, the cytoplasm of the Langerhans islet cells is stained, leaving the nuclei and the surrounding exocrine tissue unstained (Bottazzo et al., Lancet 2: 1279, 1974).
Various modifications have been introduced in attempts to improve the classic indirect immunofluorescent assay. These include use of: a) Bouin's fixed or paraffin embedded human pancreas instead of unfixed sections; b) monkey pancreas; c) acetone fixation of the tissue; d) prolonged incubation of the sera in the presence of aprotinin (a protease inhibitor); e) protein A labelled FITC as the second step reagent; f) microfluorometric apparatus to record fluorescence; g) immunohistochemical staining with glucose oxidase or peroxidase labelled protein A; h) counterstaining of the islets with monoclonal antibodies; and i) three step immunofluorescence with a biotin-avidin system (Ginsberg-Fellner and McEvoy, Autoimmunity and the Pathogenesis of Diabetes, Springer-Verlag, New York, 1990).
However, all of these assays have different degrees of specificity and sensitivity, and unknown intra- and inter-assay precision. These problems have been documented during International Workshops (1986, 1987) on the Standardization of Cytoplasmic ICA, which revealed poor inter-laboratory concordance for sera unless they were very high titer or were absolutely negative. These workshops also revealed a very broad range of end-point titers, which was ascribed to several factors but mainly related to variability in the expression of the antigenic determinants in the pancreatic tissue used in the different laboratories. The workshops have recommended preparation of an international standard ICA-positive reference serum and the establishment of common arbitrary units to express ICA results with the various methodologies employed by different laboratories. Nevertheless, it is clear that detection and quantification of islet cell antibodies by the various immunofluorescence and immunohistochemical assays is imperfect, imprecise, unstandardized, and dependent on unpredictable variables such as the freshness, source, availability, and preparation of human pancreas and the eye of the observer. As presently performed, these assays are suitable only for research projects.
The development of an immunoassay to detect and quantitate islet cell antibodies has been hampered by the lack of definition of the autoantigen(s) with which they react. A number of putative candidates have been described in the literature, including a 64 kD protein, a sialoglycolipid (ganglioside), a "polar" antigen, several other proteins of varying molecular weight, and a number of monoclonal antibody-defined antigens (Baekkeskov et al., J. Clin. Invest. 79: 926, 1987; Nayak et al., Diabetes 34: 617, 1985; Dotta et al., Clin. Res. 36: 480A, 1988; McEvoy et al., Diabetes 38: 86A, 1989). However, these substances have not been isolated in a soluble form suitable for routine clinical immunological assays. In addition, the methods used to detect the substances are not readily adaptable to a format that would be appropriate to the hospital or clinical laboratory. Thus there is a need in the art of diabetes treatment for isolated, soluble, islet cell antigens. There is also a need in the art for a method of accurately, objectively and simply determining the presence of autoantibodies that react with pancreatic islet cells.